Pulmonary epithelium is devided into three histopathologically distinct compartments: bronchi, bronchioli, and alveoli. Our long term goal is to characterize the morphological changes, aberrant cellular differentiation and genetic damage associated with progression of premalignant changes in each compartment. A. Peripheral airway cell (PAC) and neuroendocrine (NE) differentiation We have shown that field cancerization in human lung is associated with alterations in the expression patterns of PAC and NE markers. We are now developing experimental models in which the pulmonary changes parallel those seen in man. In response to carcinogen, there is a marked decrease in the expression of Clara cell specific protein (CC10), a PAC marker of progenitor cells in non-neoplastic and neoplastic airway epithelium in hamsters and mice. This is followed by NE cell hyperplasia and tumor formation later on. In order to see if reduced CC10 expression actually contributes to carcinogenesis in vivo, we exposed CC10 knockout mice to the tobacco-specific nitrosamine NNK. We observed more lung tumors in CC10 deficient animals than their wild type counterparts. This was accompanied by hyperproliferation in airway epithelium and activation of ras/MAP kinase pathway. The outcome is in concert with our in vitro experiments, in which overexpression of CC10 in lung cancer cells and immortalized bronchial epithelial cells antagonized neoplastic phenotype and affected tumor progression. Regarding NE differentiation, we were able to demonstrate that a neural transcription factor from Drosophila, achaete-schute homolog-1 is expressed in human lung and is essential for NE differentiation in neoplastic and non-neoplastic lung. Constitutive expression of this transcription factor under CC10 promoter caused marked bronchialization of alveoli, and together with SV40 massive tumors with NE differentiation. The model provides a unique tool to address the aberrant NE differentiation in the bronchiolar compartment during carcinogenesis and detailed cytogenetic and molecular characterization is under way. B. Oncogenes and tumor suppressor genes We combined PCR analyses with microdissection, and noted frequent chromosome 3p abnormalities throughout the pulmonary epithelium including BOAs and other alveolar metaplasias suggesting that these changes occur early in carcinogenesis. A novel approach using in situ PCR reaction was developed for topographic genotyping of p53 and K-ras alterations of which appear to be later events. We will now use the recently developed laser capture microdissection (LCM) to enhance our ability for more detailed topographic genotyping of the lesions in a variety of patient material, and the animal models we have created. The significance of the project is that the results will provide a rational basis for early detection and molecular targets for therapeutic interventions for human lung carcinogenesis by identifying specific markers as well as distinct models of multistep epithelial carcinogenesis.